The present invention is directed to novel amide-based cationic lipid compounds useful in lipid aggregates for the delivery of macromolecules into cells.
Lipid aggregates, such as liposomes, have been previously reported to be useful as agents for the delivery of macromolecules such as DNA, RNA, oligonucleotides, proteins, and pharmaceutical compounds into cells. In particular, lipid aggregates which include charged as well as uncharged lipids have been especially effective for delivering polyanionic molecules to cells. The reported effectiveness of cationic lipids may result from charge inmteractions with cells which are said to bear a net negative charge. It has also been postulated that the net positive charge on the cationic lipid aggregates may enable them to bind polyanions, such as nucleic acids. Lipid aggregates containing DNA have been reported to be effective agents for efficient transfection of cells.
The structure of various types of lipid aggregates vary depending on factors which include composition and methods of forming the aggregate. Lipid aggregates include, for example, liposomes, unilamellar vesicles, multilamellar vesicles, micelles and the like, and may have particle sizes in the nanometer to micrometer range. Various methods of making lipid aggregates have been reported in the art. One type of lipid aggregate comprises phospholipid containing liposomes. An important drawback to the use of this type of aggregate as a cell delivery vehicle is that the liposome has a negative charge that reduces the efficiency of binding to a negatively charged cell surface. It has been reported that positively charged liposomes that are able to bind DNA may be formed by combining cationic lipid compounds with phospholipids. These liposomes may then be utilized to transfer DNA into target cells. (See, e.g. Felgner et al., Proc. Nat. Acad. Sci. 84:7413-7417, 1987; Eppstein et al. U.S. Pat. No. 4,897,355; Feigner et al. U.S. Pat. No. 5,264,618; and Gebeyehu et al. U.S. Pat. No. 5,334,761).
Known cationic lipids include N[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium chloride (xe2x80x9cDOTMAxe2x80x9d) and combinations of DOTMA with dioleoylphosphatidylethanolamine (xe2x80x9cDOPExe2x80x9d) are commercially available. Formulation of DOTMA, either by itself or in 1:1 combination with DOPE, into liposomes by conventional techniques has been reported. However, compositions comprising DOTMA have been reported to show some toxicity to cells.
Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (xe2x80x9cDOTAPxe2x80x9d) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether, linkages to the propylamine. However, DOTAP is reported to be more readily degraded by target cells. Other cationic lipids which represent structural modifications of DOTMA and DOTAP have also been reported.
Other reported cationic lipid compounds include those which have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (xe2x80x9cDOGSxe2x80x9d) and dipalmitoyl-phosphatidylethanolamine 5-carboxyspermyl-amide (xe2x80x9cDPPESxe2x80x9d) (See, e.g. Behr et al., U.S. Pat. No. 5,171,678).
Another reported cationic lipid composition is a cationic cholesterol derivative (xe2x80x9cDC-Cholxe2x80x9d) which has been formulated into liposomes in combination with DOPE. (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). For certain cell lines, these liposomes were said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions.
Lipopolylysine, made by conjugating polylysine to DOPE has been reported to be effective for transfection in the presence of serum. (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991).
However, of the cationic lipids which have been proposed for use in delivering macromolecules to cells, no particular cationic lipid has been reported to work well with a wide variety of cell types. Since cell types differ from one another in membrane composition, different cationic lipid compositions and different types of lipid aggregates may be effective for different cell types, either due to their ability to contact and fuse with target cell membranes directly or due to different interactions with intracellular membranes or the intracellular environment. For these and other reasons, design of effective cationic lipids has largely been empirical. In addition, to content and transfer, other factors believed important include, for example, ability to form lipid aggregates suited to the intended purpose, toxicity of the composition to the target cell, stability as a carrier for the macromolecule to be delivered, and function in an in vivo environment. Thus, there remains a need for improved cationic lipids which are capable of delivering macromolecules to a wide variety cell types with greater effeciency.
In one aspect of the present invention novel amide-based cationic lipids having the structure: 
or a salt, or solvate, or enantiomers thereof are provided wherein; (a) Y is a direct link or an alkylene of 1 to about 20 carbon atoms; (b) R1 is H or a lipophilic moiety; (c) R2, R3, and R4 are positively charged moieties, or at least one but not all of R2, R3, or R4 is a positively charged moiety and the remaining are independently selected from H, an alkyl moiety of 1 to about 6 carbon atoms or a heterocyclic moiety; (d) n and p are independently selected integers from 0 to 8, such that the sum of n and o is from 1 to 16; (e) Xxe2x88x92 is an anion or polyanion and (f) m is an integer from 0 to a number equivalent to the positive charge(s) present on the lipid; provided that if Y is a direct link and the sum of n and p is 1 then one of either R3 or R4 must have an alkyl moiety of at least 10 carbon atoms.
In one embodiment R1 may be a variety of lipophilic moieties including a straight chain alkyl moiety of 1 to about 24 carbon atoms, a straight chain alkenyl moiety of 2 to about 24 carbon atoms, a symmetrical branched alkyl or alkenyl moiety of about 10 to about 50 carbon atoms, a unsymmetrical branched alkyl or alkenyl moiety of about 10 to about 50 carbon atoms, a steroidyl moiety, a amine derivative, a glyceryl derivative, or OCH(R5R6) or N(R5R6), wherein R5 and R6 are straight chain or branched alkyl moieties of about 10 to about 30 carbon.
In another embodiment when R2, R3, or R4 are positively charged moieties it is preferable that the positively charged moiety be an alkylamine moiety, a fluoroalkylamine moiety, or a perfluoroalkylamine moiety of 1 to about 6 carbon atoms, an arylamine moiety or an aralkylamine moiety of 5 to about 10 carbon atoms, a guanidinium moiety, an enamine moiety, a cyclic amine moiety, an amidine moiety, an isothiourea moiety, and a substituted heterocyclic amine moiety, a substituted heterocyclic moiety or a substituted alkyl moiety of 1 to about 6 carbon atoms substituted with a substituent selected from the group consisting of NH2, C(xe2x95x90O)NH2, NHR7, C(xe2x95x90O)NHR7, NHR7R4, or C(xe2x95x90O)NHR7R8, wherein R7 and R8 are independently selected from an alkyl moiety of 1 to about 24 carbon atoms, an alkenyl moiety of 2 to about 24 carbon atoms, an aryl moiety of about 5 to about 20 carbon atoms, and an aralkyl moiety of about 6 to about 25 carbon atoms.
When any of R2, R3, or R4 are not a positively charged moiety it is preferable that the at least one but not all of R2, R3, or R4 are independently selected from a substituted heterocyclic moiety of 1 to about 6 carbon atoms, or a substituted alkyl moiety of 1 to about 6 carbon atoms substituted with substituents selected from OH, thio, aryl of 1 to about 20 carbon atoms, or OR7, wherein R7 is an alkyl moiety of 1 to about 24 carbon atoms, an alkenyl of 2 to about 24 carbon atoms, an aryl of about 5 to about 20 carbon atoms or an aralkyl of about 6 to about 25 carbon atoms.
It is particularly prefered that when R2, R3, or R4 is an arylamine moiety that it be tryptophane, phenylanaline, or tyrosine.
In another prefered embodiment R2 is an amino acid residue having a positively charged side chain wherein the amino group(s) may be optionally substituted with an alkyl of 1 to about 6 carbon atoms or substituted to form a secondary, tertiary, or quaternary amine with an alkyl moiety of 1 to about 6 carbon atoms optionally substituted with substituents selected from hydroxyl, amino, alkoxy moiety of 1 to about 6 carbon atoms, alkylamino moiety of 1 to about 6 carbon atoms, or dialkylamino moiety of 2 to about 12 carbon atoms.
Preferably when R1 is steroidyl moiety it is a cholesteryl moiety.
It is preferable when R2 is an amino acid that it be lysine, arginine, histidine, ornithine, or an amino acid analog. In particular, when R2 is an amino acid analog it is preferable that it be 3-carboxyspermidine, 5-carboxyspermidine, 6-carboxyspermine, or monoalkyl, dialkyl, or peralkyl substituted derivatives which are substituted on one or more amine nitrogens with an alkyl group of 1 to about 6 carbon atoms.
It is prefereable that R3 and R4 independently be a lipophilic moiety of 1 to about 24 carbon atoms, a positively charged moiety, or a negatively charged moiety. In particular, when both or either R3 and R4 are a lipohilic moiety it is preferable that it be a straight chain alkyl moiety of 1 to about 24 carbon atoms, a straight chain alkenyl moiety of 2 to about 24 carbon atoms, a symmetrical branched alkyl or alkenyl moiety of about 10 to about 50 carbon atoms, a unsymmetrical branched alkyl or alkenyl moiety of about 10 to about 50 carbon atoms, an aryl moiety of about 5 to about 20 carbon atoms, an aralkyl moiety of about 6 to about 25 carbon atoms, or a steroidyl moiety.
When both or either R3 and R4 are a positively charged moiety it is preferable that the moiety be an amino acid residue having a positively charged group on the side chain, an alkylaminoalkyl moiety, a fluoroalkylaminoalkyl moiety, a perfluoroalkylaminoalkyl moiety, a guanidiniumalkyl moiety, an enaminoalkyl moiety, a cyclic aminoalkyl moiety, an amidinoalkyl moiety, an isothiourea alkyl moiety, or a heterocyclic amine moiety.
It is also preferable that when both or either R3 and R4 are a negatively charged moiety it be a carboxyalkyl moiety, a phosphonoalkyl moiety, a sulfonoalkyl moiety, or a phosphatidylalkyl moiety of 1 to about 24 carbon atoms.
It is further prefered that the sum of the integers n and p be from 1 to 8, more preferably from 1 to 4 and most preferably from 1 to 2.
It is also preferable that Xxe2x88x92 be a pharmaceutically acceptable anion or polyanion.
In a particularly prefered embodiment the amide-based cationic lipid has the structure. 
In another aspect of the present invention compositions comprising a polyanionic macromolecule and any of the lipids described above are provided. In particular, the polyanionic macromolecule may be a variety of molecules including an expression vector capable of expressing a polypeptide in a cell. In a prefered embodiment the polyanionic macromolecule is an oligomucleotide or an oligomer and most preferably DNA.
In still another aspect of the invention methods for the delivery of a polyanionic macromolecule into a cell by contacting any of the compositions above with the cell are provided. In particular, a method is provided to interfere with the expression of a protein in a cell by contacting any of the the compositions described above with a cell wherein the composition comprises an oligomer having a base sequence that is substantially complimentary to an RNA sequence in the cell that encodes the protein.
The present invention further provides a kit for delivering a polyanionic macromolecule into a cell comprising any of the compositions described above.